Src Regulates Sphingosine-1-Phosphate Mediated Smooth Muscle Cell Migration1

BackgroundSphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid released from activated platelets at sites of arterial injury that stimulates migration of smooth muscle cells (SMC). The kinase src is a significant focal point in transmembrane signaling. This study examines the role of src during smooth muscle cell migration in response to S-1-P.MethodsHuman coronary arterial SMCs were cultured in vitro. Boyden microchemotaxis assays of migration were performed in response to S-1-P in the presence and absence the src inhibitor (PP2, 10 μM) and a dominant negative src construct (DNsrc). siRNA to S-1-P receptors was used to down-regulate the S-1-P receptors. Western blotting was performed for src and MAPK phosphorylation.ResultsInhibition of src with PP2 but not PP3 partially blocked S-1-P-mediated cell migration. S-1-P induced time-dependent activation of src, which was inhibited by PP2 and adenoviral DNsrc. PP3 or an empty vector had no effect. Activation of src by S-1-P was inhibited by siRNA to S-1-PR1 and S-1-PR3 but not by S-1-PR2. When the VSMC were transfected with adenovirus containing βARKCT, an inhibitor to Gβγ, src activation was significantly attenuated. Src inhibition with PP2 reduced p38MAPK and JNK activation but did not alter ERK1/2 activation.ConclusionS-1-P mediated VSMC migration is modulated by a G-protein-coupled src pathway partially through src-mediated p38MAPK and JNK signaling and requires S-1-PR1 and S-1-PR3 receptors.